Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism

Abstract

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Rho1 activation recapitulates early gastrulation events in the ventral, but not dorsal, epithelium of Drosophila embryos

“…Rap1 and its GEF Dzy are required for proper anisotropic apical constriction and ventral furrowing, and these factors may regulate these junctional rearrangements ( Sawyer et al, 2009 ; Spahn et al, 2012 ). Ventral junctions also recruit the tension-sensitive protein Ajuba ( Winkelman et al, 2020 ; Sun et al, 2020 ; Rauskolb et al, 2019 ). These results indicate that ventral cells undergo a number of changes that might alter their response to ectopic Rho1 activation.…”

Stress fiber strain recognition by the LIM protein testin is cryptic and mediated by RhoA

“…Over the last decade, it has become more apparent that the mechanical state of filamentous actin (tension, curvature, torsion) affects the binding and activity of many actin-binding proteins (Jégou and Romet-Lemonne, 2021;Zimmermann et al, 2017). Recent work (Sun et al, 2020;Winkelman et al, 2020) has highlighted that a conserved mechanism in LIM domains confers mechanosensitivity to proteins from the FHL, zyxin and paxillin families among others to sense strained actin filaments. The LDP testin appears unique in that the FL protein resides primarily in the cytoplasm, while its N-terminal half recognizes unstrained actin filaments and its C-terminal half recognizes strained actin filaments (Figs.…”

“…2) to recognize SFSSs. Previous research has suggested that multiple LIM domains are required for mechanosensitivity(Sun et al, 2020;Winkelman et al, 2020). Surprisingly, not only did each individual LIM domain of testin display FA localization (Figs.…”

A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics

“…The pre-LIM region of Ajuba binds the Cas CCH/FAT tightly and Cas is mis-localized in ajuba-deficient cells (Pratt et al, 2005). The ajuba LIM domains bind to actin filaments under tension (Winkelman et al, 2020) and localize to adhesions (Pratt et al, 2005). Thus, ajuba or other LIM domain proteins, rather than BCAR3, may be responsible for localizing Cas to adhesions.…”

“…Zyxin binds the Cas homolog NEDD9 (Yi et al, 2002) but zyxin appears in adhesions after Cas (Zaidel-Bar et al, 2004), so is unlikely to be responsible for Cas localization. Ajuba contains a pre-LIM region that binds tightly to the Cas CCH/FATand a LIM region that binds to actin filaments under tension and localize to adhesions (Pratt et al, 2005, Winkelman et al, 2020). Moreover, Cas is mis-localized in ajuba-deficient cells (Pratt et al, 2005).…”